B7-33

Recombinant H2 relaxin (serelaxin) has gained considerable attention as a new vasoprotective drug, largely due to its potential therapeutic effects in heart failure and fibrosis. However, serelaxin is laborious and costly to produce. A single-chain peptidomimetic, B7-33, has been developed to overcome these problems but little is known about its biological actions in the vascular system. This study first compared the rapid vascular effects of an acute bolus injection of B7-33 compared with serelaxin. Male Wistar rats received a tail vein injection of placebo (20mM sodium acetate), B7-33 (13.3μg/kg) or serelaxin (26.6μg/kg). Three hours later vascular function in the mesenteric artery, small renal artery and abdominal aorta was assessed by wire myography. B7-33 and serelaxin selectively enhanced bradykinin-mediated endothelium-dependent relaxation in the rat mesenteric artery by increasing endothelium-derived hyperpolarization, but had no overall effects on relaxation in the small renal artery or aorta. We then compared the actions of B7-33 and serelaxin in an ex vivo model of vascular disease using virgin female mouse mesenteric arteries pre-incubated in placental trophoblast conditioned media to induce endothelial dysfunction characteristic of preeclampsia. Co-incubation of these arteries in trophoblast conditioned media with B7-33 or serelaxin (15, 30nM) prevented the development of endothelial dysfunction. In conclusion, equimolar doses of B7-33 replicated the acute beneficial vascular effects of serelaxin in rat mesenteric arteries and also prevented endothelial dysfunction induced by placental trophoblast conditioned media in mouse mesenteric arteries. Therefore, B7-33 should be considered as a cost-effective vasoactive therapeutic in cardiovascular diseases, including preeclampsia.

The hormone, relaxin (RLX), exerts various organ-protective effects independently of etiology. However, its complex two-chain and three disulphide bonded structure is a limitation to its preparation and affordability. Hence, a single chain-derivative of RLX, B7-33, was developed and shown to retain the anti-fibrotic effects of RLX in vitro and in vivo. Here, we determined whether B7-33 could retain the other cardioprotective effects of RLX, and also compared its therapeutic efficacy to the ACE inhibitor, perindopril. Adult male 129sv mice were subjected to isoprenaline (ISO; 25 mg/kg/day, s.c)-induced cardiomyopathy, then s.c-treated with either RLX (0.5 mg/kg/day), B7-33 (0.25 mg/kg/day; equivalent dose corrected for MW) or perindopril (1 mg/kg/day) from days 7-14 post-injury. Control mice received saline instead of ISO. Changes in animal body weight (BW) and systolic blood pressure (SBP) were measured weekly, whilst cardiomyocyte hypertrophy and measures of vascular dysfunction and rarefaction, left ventricular (LV) inflammation and fibrosis were assessed at day 14 post-injury. ISO-injured mice had significantly increased LV inflammation, cardiomyocyte hypertrophy, fibrosis, vascular rarefaction and aortic contractility in the absence of any changes in BW or SBP at day 14 post-injury. Both B7-33 and RLX equivalently reduced LV fibrosis and normalised the ISO-induced LV inflammation and cardiomyocyte hypertrophy, whilst restoring blood vessel density and aortic contractility. Comparatively, perindopril lowered SBP and the ISO-induced LV inflammation and vascular rarefaction, but not fibrosis or hypertrophy. As B7-33 retained the cardioprotective effects of RLX and provided rapid-occurring anti-fibrotic effects compared to perindopril, it could be considered as a cost-effective cardioprotective therapy.

Background Human relaxin-2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia-reperfusion injury. B7-33, a synthetically designed peptide analogous to B-chain of relaxin-2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin-2) by preferentially phosphorylating the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. We sought to investigate the effects of B7-33 treatment post ischemia-reperfusion injury in mice. Methods and Results Adult male CD1 mice were subjected to ischemia-reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7-33 significantly reduced infarct size (21.99% versus 45.32%; P=0.02) and preserved fractional shortening (29% versus 23%; P=0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7-33 and vehicle groups, respectively). In vitro, primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia-reperfusion injury (simulated ischemia reoxygenation). B7-33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7-33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP78 in an extracellular signal-regulated kinase 1/2-dependent manner. Conclusions B7-33 confers acute cardioprotection and limits myocardial infarction-related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.

BACKGROUND: Fibrosis is a hallmark of various chronic diseases, for which there is no effective cure. Whilst the recombinant form of the human peptide hormone, relaxin (RLX), is being clinically evaluated for its cardioprotective including anti-fibrotic effects in heart failure patients, this is as an injectable which is invasive. This study therefore used biodegradable nanoparticles as a delivery platform to facilitate the prolonged activity and oral application of RLX and a related mimetic as therapeutics. METHODS: RLX was conjugated to glycine-functionalised biodegradable superparamagnetic iron oxide nanoparticles (SPION-RLX), enabling therapeutic levels of RLX to be systemically or orally delivered to a murine model of cardiomyopathy. The oral (p.o) application of SPION-RLX was evaluated via daily drinking water (125 ng/5mls/day) from days 7-14 or via oral gavage every 72 h (25 ng/day) from days 14-42 post-injury. The longer-term anti-fibrotic effects of p.o administered SPION-RLX (25 ng/day) or SPION-B7-33 (25 ng/day), a single-chain RLX derivative and relaxin family peptide receptor 1 (RXFP1) agonist were compared to the frontline ACE inhibitor, perindopril (60 ng/day) from days 14-42 post-injury. RESULTS: SPION-RLX was likely phagocytosed by surveiling RXFP1-expressing dendritic cells (DCs) and transported to the circulation and target site. This allowed for the systemic or oral administration of SPION-RLX to maintain its anti-fibrotic efficacy in mice with cardiomyopathy and restore organ dysfunction after 7 days of treatment. Single-cell transcriptomics provided insights into the phagosomal uptake of SPION-RLX which may have been mediated via scavenger receptors expressed by DCs. When orally administered every 72 h to mice with established cardiomyopathy over a 4 week period, SPION-RLX or SPION-B7-33 demonstrated greater anti-fibrotic efficacy than perindopril. CONCLUSION: The conjugation of RXFP1-binding peptides to glycine-functionalised biodegradable SPIONs allowed for their circumnavigation of the gut, and prolonged activity as orally administered therapies. These findings have significant ramifications for the oral administration of peptide therapies in general.

The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) was used as a coating releasing the relaxin peptide analogue B7-33, which has been demonstrated to reduce organ fibrosis in animal models. While in vitro protein quantification was used to demonstrate controlled release of the antifibrotic peptide B7-33 from PLGA coatings, an in vitro reporter cell assay was used to demonstrate that B7-33 retains activity against the relaxin family peptide receptor 1 (RXFP1). Subcutaneous implantation of PLGA-coated polypropylene samples in mice with and without the peptide demonstrated a marked reduction in capsule thickness (49.2%) over a 6 week period. It is expected that this novel approach will open the door to a range of new and improved implantable medical devices.

Cholangiocarcinoma (CCA), the second most prevalent primary hepatic malignancy, demonstrates resistance to antiangiogenic therapy due possibly to the dynamic interaction between cancer-associated fibroblast (CAF)-mediated extracellular matrix (ECM) remodeling and angiogenesis. This study shows that anti-VEGFR2 therapy activates CAF, inducing excessive ECM deposition and forming a physical barrier that diminishes the effectiveness of antiangiogenic therapy in CCA. Based on the finding, we rationally engineered vascular endothelial cell-derived nanovesicles that inherit the angiogenic factor receptor that competitively bind and neutralize pro-angiogenic ligands to diminish their bioeffects. These nanovesicles also retain high levels of integrin αvβ3 and specifically carry peptide B7-33 (an inducer of fibroblasts quiescence) modified by the cRGD peptide, thereby developing dual-functional nanovesicles (B7-33-SNPs). The study revealed that B7-33-SNPs synergistically disrupted the CAF-angiogenesis crosstalk, effectively reducing microvessel density and fibrotic deposition in subcutaneous xenograft CCA models. This combinatorial strategy achieved a 67.7 ± 17.6% tumor growth inhibition rate through simultaneous targeting of stromal desmoplasia and vascular niches (p < 0.001 vs. PBS group). This tumor microenvironment reshaping strategy, which concurrently inhibits CAF activation and angiogenesis, offers a promising alternative for suppressing malignant CCA.